Investigating Antisense Transcription at the HTT Locus

Antisense transcription is an important mechanism of gene expression regulation. Antisense RNAs play a role in mRNA processing, translation and epigenetic modifications of DNA and histones in the locus of their origin, leading to gene silencing. HTT is a widely expressed gene, the mutation of which causes Huntington’s disease. The product of the gene plays an important role in many cell processes, such as intracellular trafficking, cell division, autophagy, and others. An antisense transcription has been found at the HTT 5’-region. The HTT-AS gene has been reported to affect HTT expression in a Dicer-dependent manner. In this study, we analyzed extensive data from RNA-seq experiments for antisense transcription at the HTT locus. Antisense transcripts corresponding to the HTT-AS gene were not found. However, we revealed a number of antisense transcripts in different parts of the locus that may take part in the regulation and functioning of the HTT gene. Keywords: antisense transcription, HTT-AS, HTTregulation, Huntington’s disease

TERT Promoter Mutations Increase Sense and Antisense Transcription from the TERT Promoter

Background: Chief among mechanisms of telomerase reverse transcriptase (TERT) reactivation is the appearance of mutations in the TERT promoter. The two main TERT promoter mutations are C>T transitions located −146C>T and −124C>T upstream from the translational start site. They generate a novel Ets/TCF binding site. Both mutations are mutually exclusive and −124C>T is strikingly overrepresented in most cancers. We investigated whether this mutational bias and mutual exclusion could be due to transcriptional constraints. Methods: We compared sense and antisense transcription of a panel of TERT promoter-luciferase vectors harboring the −124C>T and -146C>T mutations alone or together. lncRNA TAPAS levels were measured by RT-PCR. Results: Both mutations generally increased TERT transcription by 2–4-fold regardless of upstream and downstream regulatory elements. The double mutant increased transcription in an additive fashion, arguing against a direct transcriptional constraint. The −146C>T mutation, alone or in combination with −124C>T, also unleashed antisense transcription. In line with this finding, lncRNA TAPAS was higher in cells with mutated TERT promoter (T98G and U87) than in cells with wild-type promoter, suggesting that lncRNA TAPAS may balance the effect of TERT promoter mutations. Conclusions: −146C>T and −124C>T TERT promoter mutations increase TERT sense and antisense transcription, and the double mutant features higher transcription levels. Increased antisense transcription may contain TERT expression within sustainable levels.

Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1

Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.

CTCF blocks anti-sense transcription initiation at divergent gene promoters

Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is largely unknown. Here by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters, the great majority of which bear proximal CTCF binding sites. Genome editing, chromatin conformation studies, and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its chromatin architectural function. Primary transcript RNA FISH revealed co-bursting of sense and anti-sense transcripts is disfavored, suggesting CTCF-regulated competition for transcription initiation. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host’s immune response in different virus–host combinations.

Transcriptional interference in toehold switch-based RNA circuits

Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5' untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible for the ribosome. In the presence of a trigger RNA molecule protein production is turned on. Using antisense RNA against trigger molecules (anti-trigger RNA), gene expression can also be switched off again. We here study the utility and regulatory effect of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and anti-trigger transcription, whereas their cognate toehold switch, regulating expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is strongly influenced by transcriptional interference (TI) effects, leading to more than four-fold differences in expression levels. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. Based on this insight, we selected optimum promoter designs and arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers and two anti-triggers that function as a post-transcriptional RNA regulatory exclusive OR (XOR) gate.

Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences

ABSTRACTSomatic hypermutation (SHM) drives the genetic diversity of immunoglobulin (Ig) genes in activated B cells and supports the generation of antibodies with increased affinity for antigen. SHM is targeted to Ig genes by their enhancers (DIVACs; diversification activators), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase 2 (Pol2) and Pol2 occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol2 or production of full-length transcripts, indicating accumulation of stalled Pol2. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of single-stranded DNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol2 stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

Dynamics of transcription elongation are finely tuned by dozens of regulatory factors

Understanding the complex network and dynamics that regulate transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of S. cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation dynamics. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Genes that are sensitive to disruptions in transcription elongation tend to couple changes in Pol II pausing and antisense transcription to transcription output. Our findings collectively show that the regulation of transcription elongation by a diverse array of factors affects gene expression levels and co-transcriptional processing by precisely balancing Pol II activity.

There and Back Again: Hox Clusters Use Both DNA Strands

Bilaterian animals operate the clusters of Hox genes through a rich repertoire of diverse mechanisms. In this review, we will summarize and analyze the accumulated data concerning long non-coding RNAs (lncRNAs) that are transcribed from sense (coding) DNA strands of Hox clusters. It was shown that antisense regulatory RNAs control the work of Hox genes in cis and trans, participate in the establishment and maintenance of the epigenetic code of Hox loci, and can even serve as a source of regulatory peptides that switch cellular energetic metabolism. Moreover, these molecules can be considered as a force that consolidates the cluster into a single whole. We will discuss the examples of antisense transcription of Hox genes in well-studied systems (cell cultures, morphogenesis of vertebrates) and bear upon some interesting examples of antisense Hox RNAs in non-model Protostomia.